CN103649757A - A method for the identification of beta-sheet aggregated protein ligands - Google Patents
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- CN103649757A CN103649757A CN201280030461.2A CN201280030461A CN103649757A CN 103649757 A CN103649757 A CN 103649757A CN 201280030461 A CN201280030461 A CN 201280030461A CN 103649757 A CN103649757 A CN 103649757A
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Abstract
The present invention provides an assay for the identification of beta-sheet aggregated protein ligands using Thiazine Red R.
Description
The present invention relates to for differentiating the method for part, described ligand binding forms the protein of the gathering of β-pleated sheet.
In A Zihaimo patient's brain, the development of Tau protein aggregate and neurodegenerative disease is closely related, can be used as the early stage imaging mark of neurodegenerative process.Also have other neurodegenerative disease to show tau gathering.With amyloid-β imaging combination, can distinguish tau disease and A Zihaimo sick.This is for diagnosing the valuable instrument with triage.Tau imaging is the effective biomarker for tau pathology and neurodegenerative project.
Therefore, need to be used for differentiating PET(positron emission tomography) method of imaging part, described part is for the specificity imaging of the tau aggregation of A Zihaimo patient or other tau diseases.
The discovery of method of the present invention based on such,, compare the thiazin red R (Thiazine Red R) of being combined with the monomer that forms beta sheet aggregation, the thiazin red R(CAS Nr.2150-33-6 of being combined with beta sheet aggregation) show the fluorescence of strong increase.
When term " protein that beta sheet is assembled " is used in this article, finger-type becomes to have the protein of the aggregation of beta sheet structure.Tau albumen, alpha-synapse nucleoprotein and A β peptide form the aggregation with beta sheet structure.
Term " protein " refers to while using herein not relate to specific length by amino acid whose polymkeric substance.Therefore, peptide, oligopeptides and protein fragments are all included in the definition of polypeptide.Term " protein " and the interchangeable use of term " polypeptide ".
Term " Protein tau " for example refers to while using in this article, from the native sequences Protein tau of any animal (mammal, species, comprise people) and tau variant (below further definition).The separated Tau polypeptide that can (comprise people's organization type) from multiple source, or prepare Tau polypeptide by restructuring and/or synthetic method.
This mensuration can be used the Protein tau of natural or recombinant production." recombinant protein " is by allos cells and the protein of separation, purifying or discriminating, the recombinant expression carrier that described cell is expressed through transformation kinesin matter by instantaneous or stable transduction or transfection in host cell.Can for example, for example, for example, in prokaryotic (Escherichia coli (E.coli)), yeast (chestnut wine fission yeast (S.pombe)) or eukaryotic (HEK293, Sf9 insect cell) Restruction tau.Preferably, Sf9 insect cell is for the high expressed of the tau that recombinates.The Protein tau that is used for measuring can be purifying.Term " purifying " while using in this article, refer to from its natural surroundings or from recombinant production source that shift out, separate polypeptide, in described polypeptide at least 60%, more preferably at least 80% does not have their natural other relevant components, for example film and microsomes.
" native sequences tau ", no matter refer to its preparation method, has the polypeptide of the amino acid sequence identical with naturally occurring tau polypeptide.Native sequences tau can be from natural separation, or prepare by restructuring and/or synthetic method.The form naturally occurring brachymemma or secretion of tau, naturally occurring variant form (for example form of alternative splicing) and naturally occurring allelic variant specifically contained in term " native sequences tau ".In ncbi database, the identifier of people tau polypeptide is NP_005901(Seq.Id.No.1).
Term " tau variant " refers to the amino acid sequence variant of native sequences tau, contains one or more 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors and/or disappearance and/or insertion in native sequences.Amino acid sequence variant amino acid sequence general and native sequences tau has at least about 75%, preferably at least about 80%, more preferably at least about 85%, even more preferably at least about 90%, most preferably at least about 95% sequence homogeneity.
Term " A β peptide " for example refers to while using in this article, from the native sequences A β peptide of any animal (mammal, species, comprise people) and A β peptide variant (below further definition).The separated A β peptide that can (comprise people's organization type) from multiple source, or prepare A β peptide by restructuring and/or synthetic method.
This mensuration can be used the A β peptide of natural or recombinant production." recombinant protein " is by allos cells and the protein of separation, purifying or discriminating, the recombinant expression carrier that described cell is expressed in host cell with kinesin matter through transformation by instantaneous or stable transduction or transfection.Can for example, for example, for example, in prokaryotic (Escherichia coli (E.coli)), yeast (chestnut wine fission yeast (S.pombe)) or eukaryotic (HEK293, Sf9 insect cell) Restruction A β peptide.The A β peptide that is used for measuring can be purifying.Term " purifying " while using in this article, refer to from its natural surroundings or from recombinant production source that shift out, separate polypeptide, in described polypeptide at least 60%, more preferably at least 80% does not have their natural other relevant components, for example film and microsomes.
" native sequences A β peptide ", no matter refer to its preparation method, has the polypeptide of the amino acid sequence identical with naturally occurring A β peptide.It should be noted that " A β peptide " has some naturally occurring forms, thereby people's form refers to A β 39, A β 40, A β 41, A β 42 and A β 43.Topmost form A β 42 has amino acid sequence (from N end):
DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA（Seq.Id.No.3）。In A β 41, A β 40, A β 39, lack respectively C end amino acid A, IA and VIA.In A β 43 forms, at the C end of above-mentioned sequence (Seq.Id.No.3), comprise extra threonine residues.Preferred A β peptide for this mensuration is to have the amino acid sequence A β 40 that Seq.Id.No.4 provides.
Native sequences A β peptide can be from natural separation, or prepare by restructuring and/or synthetic method.Form, naturally occurring variant form and the naturally occurring allelic variant naturally occurring brachymemma or secretion of A β peptide specifically contained in term " native sequences A β peptide ".Term " A β peptide variant " refers to the amino acid sequence variant of native sequences A β peptide, and it contains one or more 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors and/or disappearance and/or insertion in native sequences.Amino acid sequence variant amino acid sequence general and native sequences A β peptide has at least about 75%, preferably at least about 80%, more preferably at least about 85%, even more preferably at least about 90%, most preferably at least about 95% sequence homogeneity.
Term " alpha-synapse nucleoprotein " for example refers to while using in this article, from the native sequences alpha-synapse nucleoprotein of any animal (mammal, species, comprise people) and alpha-synapse nucleoprotein variant (below further definition).The separated alpha-synapse nucleoprotein polypeptide that can (comprise people's organization type) from multiple source, or prepare alpha-synapse nucleoprotein polypeptide by restructuring and/or synthetic method.
This mensuration can be used the alpha-synapse nucleoprotein protein of natural or recombinant production." recombinant protein " is by allos cells and the protein of separation, purifying or discriminating, the recombinant expression carrier that described cell is expressed through transformation kinesin matter by instantaneous or stable transduction or transfection in host cell.Can for example, for example, for example, in prokaryotic (Escherichia coli (E.coli)), yeast (chestnut wine fission yeast (S.pombe)) or eukaryotic (HEK293, Sf9 insect cell) Restruction alpha-synapse nucleoprotein.Preferably, Sf9 insect cell is for the high expressed of the alpha-synapse nucleoprotein of recombinating.The alpha-synapse nucleoprotein that is used for measuring can be purifying.Term " purifying " while using in this article, refer to from its natural surroundings or from recombinant production source that shift out, separate polypeptide, in described polypeptide at least 60%, more preferably at least 80% does not have their natural other relevant components, for example film and microsomes.
" native sequences alpha-synapse nucleoprotein ", no matter refer to its preparation method, has the polypeptide of the amino acid sequence identical with naturally occurring alpha-synapse nucleoprotein polypeptide.Native sequences alpha-synapse nucleoprotein can be from natural separation, or prepare by restructuring and/or synthetic method.The form naturally occurring brachymemma or secretion of alpha-synapse nucleoprotein, naturally occurring variant form (for example form of alternative splicing) and naturally occurring allelic variant specifically contained in term " native sequences alpha-synapse nucleoprotein ".The amino acid sequence of people's alpha-synapse nucleoprotein polypeptide provides in Seq.Id.No.2.
Term " alpha-synapse nucleoprotein variant " refers to the amino acid sequence variant of native sequences alpha-synapse nucleoprotein, contains one or more 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors and/or disappearance and/or insertion in native sequences.Amino acid sequence variant amino acid sequence general and native sequences alpha-synapse nucleoprotein has at least about 75%, preferably at least about 80%, more preferably at least about 85%, even more preferably at least about 90%, most preferably at least about 95% sequence homogeneity.
The background of " test compounds " or " spike candidate compound " that term " compound " is described when relevant to mensuration of the present invention in this article.Thus, these compounds comprise organic or mineral compound, and it is from synthetic or natural origin.Compound comprises inorganic or organic compound, such as but not limited to feature, is relatively low-molecular-weight polynucleotide, lipid or hormone analogs.
Brief description
Fig. 1 has shown for saturation analysis that assemble and thiazin red R monomeric protein.On the protein of each gathering, there are 2 different binding sites for thiazin red (Thiazin-red).Determined Kd(Kd1, the Kd2 of two binding sites) A:Tau B:A β, C: Α-synapse nucleoprotein.
Experimental section
At Tris10mM pH8,24h, under the condition of 37 ℃, assembles with arachidonic acid (100 μ M) under 5 μ M concentration from the recombinant human microtubule-associated protein Tau of Escherichia coli purifying.With arachidonic acid (100 μ M), assemble synthetic A β 40, in Tris10mM pH8,37 ℃ are carried out 3 days, under 150rpm vibration.
With arachidonic acid (100 μ M), assemble people's restructuring-Α-synapse nucleoprotein of purifying from Escherichia coli, in Tris10mM pH8,37 ℃ are carried out 5 days, under 150rpm vibration.
Carry out the thiazin red saturation analysis for the protein of assembling, determine the affinity (Kd) of the protein of thiazin red and gathering.Table 1 has shown tau, the A β of thiazin red and gathering and the affinity constant of alpha-synapse nucleoprotein.Result shows, has 2 for the binding site with different affinity of thiazin red on the protein of each gathering.
The concentration of the thiazin red adding, corresponding to the Kd of the protein binding site of each gathering, replaces to induce to be added the fluorescence signal that immunomodulator compounds suppresses.
In order to determine the affinity of the thiazin red binding site of the protein of replacing immunomodulator compounds and gathering, in mensuration, add the compound of variable concentrations, scope is from 0.3nM to 10000nM.
Parallel with it, not containing under the condition of thiazin red, measure the autofluorescence of the protein of compound and gathering simultaneously.Use the protein of part and gathering as negative control, use thiazin red, there is the reference compound of known activity and the protein of gathering as positive control.
In Perkin Elmer OptiPlate384, implement to measure, in dark, 45ul measures volume, and measuring damping fluid is not containing CaCl
2and MgCl
2dPBS(GIBCO N.14020).In DMSO, dilute test compounds, add 2 μ l to (5%DMSO final concentration) in measuring.The protein of assembling by interpolation (race condition) starts to measure.Of short duration swing plate (shaking 1min with Sterico variomag teleshake), at incubated at room 30min.Under Excitation531nm/Emission595nm, use En:Vision(Perkin Elmer) measure
Table 1 has shown that tau, the A β of different compounds and gathering and alpha-synapse nucleoprotein are for the affinity constant of high affinity combined sites.
Although for the clear object of understanding, by example and the detailed description of embodiment foregoing invention, instructions and embodiment should not be regarded as having limited scope of the present invention.All patents of quoting herein and the disclosure of scientific literature are all clearly incorporated into herein by reference of text.
Claims (8)
1. for differentiating the method for the protein ligands that beta sheet is assembled, comprising:
A) protein that beta sheet is assembled contacts with compound with thiazin red R,
B) the thiazin red R fluorescence in measuring process potpourri a), wherein the thiazin red R fluorescence in the potpourri of step a) is compared blank minimizing, the protein ligands of having indicated beta sheet to assemble.
2. the process of claim 1 wherein that the protein that beta sheet is assembled is selected from Protein tau, A β peptide and alpha-synapse nucleoprotein.
3. claim 1 or 2 method, the protein that wherein beta sheet is assembled is Protein tau or A β 40 peptides, preferably people's Protein tau or people A β 40 peptides.
4. the method for claims 1 to 3, wherein thiazin red R fluorescence is measured at Excitation531nm/Emission595nm.
5. the method for claim 1 to 4, the protein that wherein beta sheet is assembled is the protein that the beta sheet of recombinant production is assembled.
6. the method for claim 1 to 5, its empty comprises protein and the thiazin red R that beta sheet is assembled.
7. the method for claim 1 to 6, wherein compound is that the concentration that increases is added in the potpourri of step a), thereby determines the IC50 of test compounds.
8. thiazin red R is for differentiating the purposes of the protein ligands that beta sheet is assembled.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP11170878 | 2011-06-22 | ||
| EP11170878.0 | 2011-06-22 | ||
| PCT/EP2012/061621 WO2012175458A1 (en) | 2011-06-22 | 2012-06-19 | A method for the identification of beta-sheet aggregated protein ligands |
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| Publication Number | Publication Date |
|---|---|
| CN103649757A true CN103649757A (en) | 2014-03-19 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201280030461.2A Pending CN103649757A (en) | 2011-06-22 | 2012-06-19 | A method for the identification of beta-sheet aggregated protein ligands |
Country Status (10)
| Country | Link |
|---|---|
| US (1) | US20140363898A1 (en) |
| EP (1) | EP2724161A1 (en) |
| JP (1) | JP2014520270A (en) |
| KR (1) | KR20140026565A (en) |
| CN (1) | CN103649757A (en) |
| BR (1) | BR112013031498A2 (en) |
| CA (1) | CA2837957A1 (en) |
| MX (1) | MX2013014006A (en) |
| RU (1) | RU2013158623A (en) |
| WO (1) | WO2012175458A1 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11254718B2 (en) | 2019-09-04 | 2022-02-22 | Amprion, Inc. | Alpha-synuclein substrates and methods for making and using the same |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2004252182A1 (en) * | 2003-06-27 | 2005-01-06 | Proteome Systems Ltd | Method of isolating a protein |
| CN1780613A (en) * | 2003-02-27 | 2006-05-31 | 乔安妮·麦克劳林 | Methods of preventing,treating and diagnosing disorders of protein aggregation |
-
2012
- 2012-06-19 KR KR1020137034019A patent/KR20140026565A/en not_active Application Discontinuation
- 2012-06-19 MX MX2013014006A patent/MX2013014006A/en not_active Application Discontinuation
- 2012-06-19 BR BR112013031498A patent/BR112013031498A2/en not_active Application Discontinuation
- 2012-06-19 EP EP12728527.8A patent/EP2724161A1/en not_active Withdrawn
- 2012-06-19 WO PCT/EP2012/061621 patent/WO2012175458A1/en active Application Filing
- 2012-06-19 JP JP2014516297A patent/JP2014520270A/en active Pending
- 2012-06-19 RU RU2013158623/15A patent/RU2013158623A/en unknown
- 2012-06-19 CA CA2837957A patent/CA2837957A1/en not_active Abandoned
- 2012-06-19 CN CN201280030461.2A patent/CN103649757A/en active Pending
-
2013
- 2013-12-20 US US14/137,399 patent/US20140363898A1/en not_active Abandoned
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1780613A (en) * | 2003-02-27 | 2006-05-31 | 乔安妮·麦克劳林 | Methods of preventing,treating and diagnosing disorders of protein aggregation |
| AU2004252182A1 (en) * | 2003-06-27 | 2005-01-06 | Proteome Systems Ltd | Method of isolating a protein |
Non-Patent Citations (3)
| Title |
|---|
| EMILIA BRAMANTI ET AL: "Effects of hypericin on the structure and aggregation properties of beta-amyloid peptides", 《EUROPEAN BIOPHYSICS JOURNAL》, vol. 39, no. 11, 15 May 2010 (2010-05-15) * |
| HONSON ET AL: "Differentiating Alzheimer disease-associated aggregates with small molecules", 《NEUROBIOLOGY OF DISEASE》, vol. 28, no. 3, 19 November 2007 (2007-11-19), XP022350875, DOI: doi:10.1016/j.nbd.2007.07.018 * |
| MENA RAUL ET AL: "Monitoring pathological assembly of tau and beta-amyloid proteins in Alzheimer"s disease", 《ACTA NEUROPATHOLOGICA》, vol. 89, no. 1, 1 January 1995 (1995-01-01), XP009162241 * |
Also Published As
| Publication number | Publication date |
|---|---|
| MX2013014006A (en) | 2014-03-12 |
| KR20140026565A (en) | 2014-03-05 |
| JP2014520270A (en) | 2014-08-21 |
| EP2724161A1 (en) | 2014-04-30 |
| US20140363898A1 (en) | 2014-12-11 |
| RU2013158623A (en) | 2015-07-27 |
| CA2837957A1 (en) | 2012-12-27 |
| BR112013031498A2 (en) | 2017-05-30 |
| WO2012175458A1 (en) | 2012-12-27 |
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